Environ Biol Fish https://doi.org/10.1007/s10641-019-00897-0 Feeding habits and ecological role of the freshwater stingray Potamotrygon magdalenae (Duméril 1865) (Myliobatiformes: Potamotrygonidae), combining gut-content and stable isotope analysis Viviana Márquez-Velásquez & Ricardo S. Rosa & Esteban Galindo & Andrés F. Navia Received: 21 February 2019 / Accepted: 20 June 2019 # Springer Nature B.V. 2019 Abstract Understanding the ecological role of a species in an ecosystem and the dynamics of the communities depends largely on knowledge of the trophic relationships. We evaluated the feeding habits and the trophic ecology of the endemic Colombian stingray Potamotrygon magdalenae, integrating stomach content and isotopic analyses (13C and 15N). The samples were collected in the middle Magdalena River basin, Colombia, during artisanal fishing operations in the dry and rainy seasons. The stomach content analysis indicated that P. magdalenae fed on a high number of occasional items, such as seeds, Planariidae, Teleostei and Nematoda, with Diptera being the dominant food component at the population level. There were no significant differences in diet between males and females. In contrast, isotopic analysis showed that Coleoptera was the most important food source assimilated by the species, followed by Ephemeroptera; Chironomidae and Trichoptera made the lowest contributions. No significant differences in δ13C and δ15N were observed between the sexes or hydrological seasons. Estimates of the isotopic niche indicated that P. magdalenae has a narrower trophic niche than the teleost fishes present in the study area. The trophic level was identified as intermediate, suggesting that Potamotrygon magdalenae plays a role as a mesopredator in the food web in the study area. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10641-019-00897-0) contains supplementary material, which is available to authorized users. Introduction V. Márquez-Velásquez (*) Programa de Pós-Graduação em Ciências Biológicas (Zoologia), Universidade Federal da Paraíba, João Pessoa, PB 58059-900, Brazil e-mail: vmarquez@squalus.org V. Márquez-Velásquez : E. Galindo : A. F. Navia Fundación Colombiana para la Investigación y Conservación de Tiburones y Rayas, SQUALUS, Calle 10ª No. 72-35, Apto. 310E, Cali, Colombia R. S. Rosa Departamento de Sistemática e Ecologia, Centro de Ciências Exatas e da Natureza, Universidade Federal da Paraíba, João Pessoa, PB 58051-900, Brazil Keywords Batoidea . Diet . Feeding sources . Food webs . δ13C and δ15N Knowledge and understanding of the feeding ecology of species provides fundamental information on community dynamics and the functional role that species play in the structure of ecosystems (Braga et al. 2012). The recent advances in studies on elasmobranch diets have shown a shift away from broad generalizations characterizing all elasmobranchs as apex predators to more quantitative multispecies dietary assessments (Cortés 1999; Ebert and Bizzarro 2007; Navia et al. 2017). Specifically, rays (Batoidea) are considered to have an important role in energy transfer in the trophic networks of benthic and demersal marine ecosystems (Ebert and Bizzarro 2007; Wetherbee et al. 2012; Jacobsen and Bennett 2013). Environ Biol Fish Rays of the family Potamotrygonidae are important components of the Neotropical ichthyofauna and are strongly threatened by anthropogenic actions, such as ornamental trade, habitat degradation, and dam construction. They are the only extant group of elasmobranchs adapted to living exclusively in freshwater environments (Compagno and Cook 1995) and are widely distributed in several river basins in South America (Rosa et al. 2010). There are four freshwater genera and 31 freshwater species (Lasso et al. 2016). Two marine species previously described in the the amphi-American genus Himantura (H. scharmardae and H. pacifica), were relocated in the recently established genus Styracura (Carvalho et al. 2016), and included in this family (Carvalho et al. 2016). Like most marine batoids, freshwater rays are carnivorous species and feed on a large variety of prey (Shibuya et al. 2009). Specialized food habits have been identified in some species, such as Potamotrygon orbignyi (Castelnau, 1855) and P. signata (Garman, 1913) (Moro et al. 2011, 2012), and feeding plasticity has been identified in others, such as P. motoro (Müller and Henle 1841) (Lonardoni et al. 2006; Silva and Uieda 2007; Lasso et al. 2013). Knowledge regarding the diet of the South American freshwater stingrays has increased considerably over the last few years, especially in the species distributed in Brazil (e.g., Shibuya et al. 2009; Gama and Rosa 2015). Despite the several studies evaluating the ecological role of freshwater fishes in Neotropical aquatic food webs (e.g., Lowe-McConnell 1987; Jepsen and Winemiller 2002), feeding differentiation among species (Mérona and Rankin-de-Mérona 2004; Correa and Winemiller 2014), and how these roles shift in response to changes in environmental conditions, ontogenetic stage or even sex (Werner and Gilliam 1984; Junk et al. 1989; Winemiller 1989; Winemiller and Jepsen 1998; Keppeler et al. 2015), none have included species of Potamotrygonidae. Additional similar studies are essential to understand how food resources affect their distribution, abundance and coexistence and to evaluate in detail the ecological importance of these species in freshwater ecosystems (Lowe-McConnell 1987; Abelha et al. 2001; Arrington and Winemiller 2004). Potamotrygon magdalenae is an endemic freshwater stingray species from the Magdalena River basin in Colombia. Like its congeners, its capture for ornamental purposes is one of the main threats, currently being the most exported ornamental fish in Colombia and eventually used as food by fishermen communities (Mojica et al. 2012). It is impacted by several anthropogenic activities, such as water pollution, mining activities, deforestation and dam construction (Mojica et al. 2012). Freshwater stingrays are more susceptible to impacts and exhibit lower tolerance to both natural and anthropogenic impacts than their marine counterparts due to their more restricted distribution (Charvet-Almeida et al. 2002). Accordingly, considering its fishing pressure, trade and restricted distribution, P. magdalenae has been included as a very high priority species in the National Action Plan for the conservation and management of sharks, rays and chimaeras of Colombia (Caldas et al. 2010), and thus baseline studies to acquire more information on its life history aspects are needed. The only previous study on its feeding habits, based solely on stomach contents, was carried out in the lower Magdalena River, suggesting that P. magdalenae is a specialist predator, exploiting mainly larval and adult insects but also consuming mollusks and detritus (RamosSocha and Grijalba-Bendeck 2011). However, there are still unknown aspects regarding the variation in its feeding habits according to hydrological period, sex and developmental stage and its topological role in trophic networks. Stable isotopes of nitrogen (δ15N) and carbon (δ13C) have been used to complement stomach content analysis in the study of the trophic ecology of elasmobranch species, especially in marine ecosystems (e.g., Hussey et al. 2012), with only one study so far in freshwater elasmobranchs (MacNeil et al. 2006). The use of multiple approaches allows more robust assertions about the trophic patterns and trophic interactions among species (e.g., Weidner et al. 2017). In this study, we aim i) to investigate the feeding habits and trophic ecology of the endemic stingray Potamotrygon magdalenae in the Magdalena River basin, Colombia, integrating stomach content and isotopic analysis (13C and 15N) techniques, ii) to evaluate the influence of sex and hydrological season on the δ13C and δ15N signals for P. magdalenae and iii) to compare the isotopic niches between P. magdalenae and coexisting bony fish species, providing new insights into the ecological role of this endangered batoid species in the Magdalena River. Environ Biol Fish Methods Study area The Magdalena River is the largest fluvial system in Colombia; it is 1612 km in length and originates from headwaters in the Andean Cordillera at an elevation of 3300 m. The Magdalena basin supports 220 fish species (DoNascimiento et al. 2017) and includes the most productive fishing areas in Colombia (Galvis and Mojica 2007). The study area is the mid-course of the Magdalena River basin in the municipalities of Puerto Boyacá and Puerto Nare between the Antioquia and Boyacá departments (6°04′55”N-74°34′00”W, 6°21′50”N-74°29′50”W; 125 m elevation) and is located within the Tropical Moist Forest life zone (Holdridge 1987). This region includes extensive lowland alluvial plains with many marginal lakes covering an estimated area of 22,000 km2 (Garzón and Gutiérrez 2013), which constitute temporary or permanent habitats for communities of phytoplankton, zooplankton, macrophytes, insects, amphibians, birds and mammals (Moreno and Fonseca 1987) and represent an important habitat for the local ichthyofauna. The climate is characterized by a bimodal rainfall regime. December–March and June–September are dry periods, and April–May and October–November are rainy periods (IDEAM 2015, 2016). The two wet seasons are comparable in length and intensity. The mean annual precipitation is 2917 mm, the mean annual temperature is 27.9 °C, and the relative humidity is 80% (IDEAM 2016). Human activities, such as hydroelectric dam construction, cattle raising and oil exploitation, have historically modified the middle Magdalena valley (Garzón and Gutiérrez 2013). Consequently, this study region is a land mosaic formed by human settlements, small and highly disturbed native vegetation patches and extensive areas of pasture and oil infrastructure. Sampling The stingrays were collected during both the dry (June– July and December) and rainy (October and November) seasons in 2015 and 2016 as the bycatch of a local fishing boat. Each individual of P. magdalenae was weighed in g (M) and measured in cm [disc width (DW)], and the sex was determined by the presence or absence of claspers. Stomachs were removed from each specimen, labeled and preserved in 10% formaldehyde. Muscle tissue samples (2 cm3) were taken from each specimen, labeled, stored in plastic bags and preserved in ice for transportation to the laboratory, where they were frozen at −20 °C and stored until processing. Additionally, we sampled bony fish (also caught by local fishermen) and invertebrate species during the same periods and in the same fishing zones (Electronic Supplementary Material Table S1). Invertebrates represent potential prey items and were sampled from the riparian leaf litter in shallow areas using a dip net (Paetzold et al. 2005). These samples were similarly preserved in ice and frozen at −20 °C and stored until processing. Stomach content analysis In the laboratory, each stomach was dissected, and prey items were identified to the lowest taxonomic level possible. Prey items were identified with identification keys for invertebrates (Roldán 1988; Roldán et al. 2014). After taxonomic identification, the dietary items were counted and weighed on an analytical balance (0.001 g). To determine whether the number of analyzed stomachs accurately described the diet of P. magdalenae, a prey accumulation curve was created for the species and for females and males separately. The samples were randomized 50 times with the routine “sample-based rarefaction” in the EstimateS 7.5 software (Colwell 2005) following Cortés (1997). Accuracy was evaluated by comparing the slope of the last four points of the curve with a zero-slope line using Student’s t test with a 95% confidence level (p > 0.05; Bizzarro et al. 2007). The average coefficient of variation (CV) of the last points was calculated as CV = standard deviation*100 /average (Bizzarro et al. 2007) to obtain a measure of precision. The contribution of each prey item to the diet of P. magdalenae was estimated using two indices adjusted to the prey-specific abundance (in terms of percent number (%PNi) and percent weight (%PWi)) in addition to the percent frequency of occurrence (%FO). The prey-specific abundance (%PAi) was calculated following Brown et al. (2012): %PAI ¼ ∑nj¼1 %Aij ni where %Aij is the abundance (by count (%PNi) or weight (%PWi)) of prey category I in stomach sample j and ni is the number of stomachs containing prey i. The index of relative importance (IRI) was modified and replaced with the prey-specific index of relative Environ Biol Fish importance (PSIRI) to incorporate the prey-specific abundance measures (%PNi and %PWi) in the calculations (see Brown et al. 2012): %PSIRI ¼ %FOi  ð%PN i þ %PW i Þ 2 The feeding strategy of the species was described using the graphical method proposed by Costello (1990) and modified by Amundsen et al. (1996). This analysis allows us to infer whether the predator is a specialist or generalist at the individual or population level. It relates the frequency of occurrence of a given prey type with the prey-specific abundance (PAi), which is defined in this case as the total weight of prey i divided by the total weight of the contents of the stomachs containing prey i (Costello 1990; Amundsen et al. 1996). Prey with a high specific abundance and low frequency of occurrence (upper left of the graph) are eaten by a few individuals displaying individual specialization, whereas prey with a low specific abundance and high occurrence (lower right of the graph) are eaten occasionally by most individuals. Prey with a high specific abundance and high frequency of occurrence (upper right of the graph) represent specialization of the predator population, whereas prey with a low specific abundance and low frequency of occurrence (lower left of the graph) represent rare prey consumed by few individuals (Amundsen et al. 1996). The niche width was estimated using Levin’s standardized index: Bi = (1/n-1) × 1/ΣPij2–1, where Pij is the proportion of predator diet i that is made up of prey j, and n is the number of prey categories (Krebs 1999). This index was calculated by applying the %PN values (converted to proportions) of the different dietary items identified. This index ranges from 0 to 1; zero means that a species consumed only one type of prey, whereas one means that a species ingested many types of prey. The mass percentages (%M) of each identified prey item were used to evaluate whether the diet of the species differed between sexes. Each prey’s weight contribution was root square transformed, and a similarity matrix was constructed using the Bray-Curtis similarity coefficient. A nonmetric multidimensional scaling analysis (nMDS) was used to evaluate similarity (Clarke 1993). Subsequently, ANOSIM was carried out to test the significance of the observed patterns in the nMDS. The R statistic allows the evaluation of similarity between groups, with values close to 0 indicating a lack of difference, and values close to −1 and 1 indicating differences in diet. The P values generated from the R statistic were considered significant when p < 0.05. All analyses were carried out using PRIMER v6 software (Clarke and Gorley 2006). To carry out multivariate analyses, the data were prepared as follows: 1) Stomachs were randomly distributed into groups of six to 10 and separated by sex, 2) an average abundance value (%M) was calculated for each of the feeding categories to reduce the number of prey categories with zero values and increase the effectiveness of the multivariate analyses (White et al. 2004), 3) average %M values for the grouped data calculated for each prey category were square root transformed and used to construct a similarity matrix with Bray-Curtis similarity values (Platell et al. 1998). The trophic level of each species (TL) was calculated following Cortés (1999): ! T Li ¼ 1 þ n ∑ Pj  TLj j¼1 where Pj is the proportion that each prey category represents in the diet of the predator and TL j is the trophic level of each prey category, j. The trophic levels of prey categories at the family, genus, or species level were taken from Vander Zanden et al. (1997). Stable isotope analysis Each tissue sample was rinsed in distilled water, placed in sterile Petri dishes and dried in an oven at 60 °C for 48–72 h. Lipids were extracted from tissue samples of P. magdalenae and bony fishes with a 2:1 chloroformmethanol solution (Folch et al. 1957; Bligh and Dyer 1959). Afterward, the tissue samples were rinsed with deionized water to remove nitrogenous compounds. Although there is evidence that lipid extraction might influence isotopic values (Sotiropoulos et al. 2004; Murry et al. 2006; Logan et al. 2008), these techniques are advantageous because they remove the majority of lipids, standardizing data among species within a food web (Post et al. 2007). Individuals were pooled across each invertebrate taxon to ensure sufficient biomass for isotopic analysis, then lyophilized whole and homogenized. Dried samples were ground to a fine powder with a glass mortar and pestle and stored in glass containers. Subsamples were weighed (∼1 mg for animal tissues) and pressed into ultra-pure tin capsules. This material Environ Biol Fish was sent to the Stable Isotope Facility at the University of California-Davis, USA, for determination of the isotopic ratio. C and N stable isotope ratios were measured using a PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20–20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK). The results are expressed in delta notation (parts per thousand deviation from a standard material): δ13C or δ15N = [( Rsample/Rstandard) − 1] × 1000, where R = 13C/12C or 15 N/14N. The R standard values were based on the Vienna Peedee belemnite (VPDB) for δ13C and atmospheric nitrogen for δ15N. Student’s t-tests were used to assess differences in δ13C and δ15N between sexes and hydrological seasons. The isotopic niche and trophic overlap of females and males of P. magdalenae in the two hydrological seasons were estimated by Bayesian standard ellipse corrected areas (SEAC, expressed in ‰2, adjusted for small sample size) using the SIBER package (Jackson et al. 2011). Additionally, the isotopic niche of the other species of fishes was estimated, but only for the dry season due the small sample sizes. The species were classified into trophic guilds according to information provided by the online database FishBase (Froese and Pauly 2018). Unlike other methods for estimating these trophic parameters (e.g., convex hull; Layman et al. 2007), SEAC estimates are less susceptible to outliers (Jackson et al. 2011; Syväranta et al. 2013). All statistical analyses were carried out in R software (version 3.4.3; R Core Team 2017). The parameters used to calculate and plot the SEA included the angle in radians (θ) between a and the xaxis and the eccentricity (E) of the standard ellipse, which is determined by the variance along the x- and y-axes as 0 < E < 1, where E = 0 is a perfect circle and E close to 1 indicates that the standard ellipse is more elongated (Jackson et al. 2011; Parnell and Jackson 2011). θ is returned as a value between 0 and π and is reported here in degrees between 0° and 180°, where positive or negative values indicate the inclination of the ellipse. θ values close to 0° represent relative dispersion along the x-axis (δ13C), indicating utilization of multiple resources or source mixing, whereas as θ values approach 90°, the ellipse is dispersed along the y-axis (δ15N), indicating that individuals within a site are feeding across different trophic positions within a uniform basal source (Jackson et al. 2011). The proportional contribution of each prey item to the diet of P. magdalenae was inferred with Bayesian stable isotope mixing models (Parnell et al. 2013) using the MixSIAR package (Stock and Semmens 2013) in R software. The MixSIAR model uses variation in isotopic discrimination factors and Bayesian inference to produce the most likely set of proportional contributions of sources for a given consumer. Before running the model, the point-in-polygon assumption was evaluated using the Monte Carlo simulation of mixing polygons proposed by Smith et al. (2013). This method considers uncertainty in trophic enrichment factors and sources, providing a quantitative measure of the proposed mixing model suitability. Using this approach, eleven consumers were excluded from the MixSIAR analysis (those that were outside of the 95% mixing region). We used average values obtained from previous meta-analyses, with trophic fractionation of δ13C = 0.5 ± 0.13 SD (McCutchan et al. 2003) and trophic fractionation of δ15N = 2.5 ± 0.11 SD (Vanderklift and Ponsard 2003). Additionally, the information obtained from the stomach content analysis was used as informative priors to refine the Bayesian mixing model. The model was fitted with three Markov chain Monte Carlo simulations (length: 300,000, burn-in: 200,000, thin: 100), and the convergence assumption was assessed with Gelman-Rubin diagnostics (Gelman et al. 2014). Nitrogen isotopic signatures were used to estimate the trophic positions (TPs) of P. magdalenae and the other consumers according to the following equation proposed by Post (2002): TP ¼ λ þ δ15N Predador − δ15N Base ; △n where λ is the trophic position of the baseline organism, periphyton (λ = 1); δ15NBase is the mean δ15N of this primary producer in the system (4.95‰), and △n=2.54 ‰ is the mean trophic fractionation value found in a previous meta-analysis (Vanderklift and Ponsard 2003). Results Stomach content analysis A total of 74 stomachs were analyzed from individuals measuring 7.50 to 22.25 cm DW, including 41 females (7.50 to 22.50 cm DW; mean = 14.14 ± 3.96 SD), 30 males (7.60 to 21 cm DW; 13.36 ± 2.79) and three individuals of unidentified sex. Of the total, 59 Environ Biol Fish individuals were juveniles and 12 were adults. Due to the unbalanced number of these two ontogenetic categories, which could cause statistical bias, they were not treated separately in the diet analyses. Of the analyzed stomachs, three (4.05%) stomachs were empty, and a total of 45 food components (prey items) were identified and grouped into 12 major prey categories (Table 1). The cumulative diversity curve for the species reached an asymptote (p > 0.05), and the coefficients of variation were < 0.01 (Fig. 1), indicating that the number of analyzed stomachs accurately described the diet of P. magdalenae but not separately for males and females or juveniles and adults (p < 0.05). The most frequent prey categories were Diptera (76.0%) and Trichoptera (23.9%). Chironomidae larvae and Ceratopogonidae larvae were the most frequent prey items (64.8% and 43.6%, respectively) (Table 1). Unidentified insect parts also showed a high frequency in the diet of P. magdalenae (50.7%). The most important prey categories according to prey-specific abundance by number (%P N ) were Bivalvia and Diptera larvae (53.6% and 50.6%, respectively) and by mass (%P M ) were Bivalvia and Oligochaeta (99.7% and 45.8%, respectively). Unidentified insect parts had a significant value for both indices (77.7% and 72.5%) (Table 1). The results of the % PSIRI indicated that the diet of P. magdalenae is based on insects, especially Chironomidae larvae (23.2%). Ceratopogonidae larvae were the second most important (8.4%), followed by Oligochaeta (5.7%). At highest taxonomic level, Diptera and unidentified insects represented the dominant categories in the species’ diet (Table 1). The graphical analysis of the feeding strategy showed that individuals of P. magdalenae fed on a high number of occasional items, such as seeds (Se), Planariidae (Pl), Teleostei (Te), Nematoda (Ne) and Diplostraca (Dp). Unidentified insect parts (In) and Diptera (Di) were the dominant food components at the population level (Fig. 2a), indicating a tendency to dietary specialization. This pattern was also observed separately in males and females, where males showed a higher frequency and abundance of unidentified insects than females (Fig. 2b, c). The categories Oligochaeta (Ol), Diptera (Di), Ephemeroptera (Ep) and Mollusca (Mo) were more abundant among females. There were no significant differences in diet between males and females (R = 0.05; p = 0.31, Fig. 2d). Table 1 Percent frequency of occurrence (%FO), prey-specific abundance by number (%PN), prey-specific abundance by mass (%PM), and prey-specific index of relative importance (%PSIRI) for Potamotrygon magdalenae prey items in the middle Magdalena River basin. Source origin: All: Allochthone, Aut: Autochtone Prey categories % FO %PN %PM %PSIRI Origin Planariidae 7.04 11.52 16.69 0.99 Aut Phylum Nematoda 4.22 3.62 0.11 Aut Phylum Mollusca 4.22 21.13 34.51 1.17 Bivalvia 1.41 53.57 99.75 1.08 Platyhelminthes 1.66 Gastropoda Planorbidae Aut Aut 1.41 4.91 1.89 0.05 Aut 2.82 1.31 0.83 0.03 Aut 15.49 28.13 45.82 5.73 Aut Trichoptera 23.94 13.00 14.61 3.31 Glossosomatidae larvae Leptoceridae larvae 1.41 1.43 0.02 4.22 5.50 10.14 0.33 Aut Atanatolica sp. 1.41 9.30 4.49 0.10 Aut Hydropsychidae larvae 1.41 10.00 5.53 0.11 Aut Trichoptera larvae 14.14 15.85 2.75 Aut Crustacea Diplostraca Annelida Oligochaeta Insecta 18.31 1.94 Aut Coleoptera 26.76 5.34 5.39 1.44 Elmidae 9.86 2.33 2.72 0.25 Aut Macrelmis sp. 4.22 3.88 0.49 0.09 Aut Hydrophilidae 2.82 3.08 0.33 0.05 All-Aut Staphylinidae 1.41 1.92 0.82 0.02 All Ptilodactylidae 5.63 1.28 4.68 0.17 All Coleoptera n.i. 12.68 6.71 6.86 0.86 Diptera 76.06 56.73 53.26 41.83 Ceratopogonidae larvae Chironomidae larvae 43.66 20.07 18.61 8.44 64.79 35.39 36.37 23.25 Aut Chironomidae Pupae 16.90 10.80 13.27 2.03 Aut Aut Dolychopodidae larvae 1.41 2.27 0.23 0.02 Aut Empididae larvae 5.63 3.47 6.43 0.28 Aut Lispe sp. 1.41 1.43 0.92 0.02 Aut Diptera larvae 18.31 50.64 33.79 7.73 Aut Diptera pupae 1.41 8.00 0.06 Aut Muscidae larvae Aut 0.62 Ephemeroptera 22.53 23.08 27.199 5.66 Euthyplociidae 1.41 10.46 22.40 0.23 All-Aut Euthyplocia sp. 1.41 47.06 27.14 0.52 Aut Leptophlebiidae 1.41 0.33 16.15 0.12 All-Aut Terpides sp. 1.41 4.28 1.38 0.04 Aut Thraulodes sp. 2.82 28.47 32.87 0.86 Aut Traverella sp. 4.22 10.20 2.31 0.26 Aut Environ Biol Fish Table 1 (continued) Stable isotope analysis % FO %PN %PM %PSIRI Origin Oligoneuriidae 1.41 0.13 Lachlania sp. 1.41 7.14 4.61 0.08 Aut Polymitarcyidae larvae 2.82 2.11 15.69 0.25 Aut Aut Prey categories 7.95 10.73 Ephemeroptera larvae 16.90 16.69 20.72 3.16 Insect remains 50.70 77.66 72.47 38.06 Insects n.i 43.24 89.38 80.78 37.15 Aut Hemiptera Notonectidae 1.41 0.24 0.75 0.01 Hemiptera n.i. 1.41 0.71 10.50 0.08 Aut Formicidae 1.41 0.89 2.50 0.02 All Lepidoptera Larvae 2.82 1.29 1.01 0.03 All Libellulidae 1.41 1.43 0.37 0.01 Aut Odonata Larvae 5.63 4.78 22.07 0.76 Aut Plecoptera nymphs 1.41 0.02 0.38 0.00 Aut Teleostei n.i. 4.22 3.07 10.11 0.28 Aut Seeds 7.04 19.09 20.10 1.38 All Hymenoptera Odonata Teleostei The major prey categories are indicated in bold The calculated niche width of P. magdalenae when including 45 prey items was 0.08, whereas it was 0.22 when including 12 prey categories, both results indicating that the species is a specialist, with marked preferences for Diptera (Fig. 2a–c). Females showed a broader trophic niche than males. The P. magdalenae trophic level was intermediate (TL = 3.50), and its role in the ecosystem seems to not be influenced by sex (Fig. 2b, c). 2.0 Shannon - wienner index Fig. 1 Cumulative prey curves for Potamotrygon magdalenae. The solid black line is the cumulative prey diversity index and the dashed lines are the standard deviation A total of 24 samples of P. magdalenae muscle were collected, 12 in the rainy season and 12 in the dry season. In the rainy season, females and males were depleted in δ13C ratios and had similar δ15N ratios, whereas in the dry season, the δ15N ratios were similar between the sexes (Table 2). However, there were no significant differences between the sexes for δ13C (ttest, t = −0.02, p = 0.99) or δ15N (t = −0.94, p = 0.36) or between hydrological seasons (δ13C: F = 0.12, p = 0.73; δ15N: F = 0.21, p = 0.65). Females and males were not as clearly differentiated in isotopic space based on the δ13C and δ15N values in both the rainy and dry seasons (Fig. 3a). However, the overlap between the sexes according to hydrological season ranged from low overlap within the rainy season to 80% overlap within the dry season (Table 2). The parameter E was similarly high between females and males (> 0.97), indicating an elongated ellipse. Additionally, the parameter θ showed a more relative dispersion along the x-axis (δ13C) for both sexes, indicating the utilization of multiple resources or source mixing (Table 2, Fig. 3a). Among the 97 samples of the 16 bony fish species, δ15N ranged from 7.76‰ to 11.76‰, and δ13C ranged from −31.63‰ to −21.17‰. In the 22 samples of insects, including the potential prey items [Ephemeroptera, Trichoptera, Diptera (Chironomidae), Coleoptera (Elmidae)], δ15N ranged from 5.7‰ to 11.3‰, and δ13C ranged from −25.7‰ to −23.5‰ (Table 3). 1.5 1.0 0.5 T-student = 1.732, p> 0.05 0.0 0 10 20 30 40 50 Number of stomachs 60 70 Environ Biol Fish 100 (Bi=0.08; TL=3.50) a) (Bi=0.04; TL=3.49) b) 80 In In Pi (%) 60 Di Ol 40 Ol Di Mo Ep 20 0 Se Pl Te Dp Ne 0.2 0 100 Tri Co 0.4 Tri Ep Pl Te Dp Mo Se Co Ne Species 0.6 0.8 1 (Bi=0.17; TL=3.49) c) Pi (%) 40 0.4 0.6 0.8 1 2D Stress=0.14 d) Males 80 60 0.2 0 Males In Mo Se Pl Di Ol Ep Females 20 Ne Dp 0 0 0.2 Tri Co 0.4 0.6 Frequency Females 0.8 1 Fig. 2 Graphical analysis of the species for Potamotrygon magdalenae (a) and its respective categories (b = males, c = females) with each of their groups of prey (Co) Coleoptera, (Dp) Diplostraca, (Di) Diptera, (Ep) Ephemeroptera, (Mo) Mollusca, (Ne) Nematoda, (Ol) Oligochaeta, (Te) Teleostei, (Pl) Planariidae, (In) Insects, (Se) Seeds, (Tri) Trichoptera. d. Non-metric multidimensional scaling ordination plot of mean gravimetric dietary data (per cent mass (%M)) for females (□) and males (▲) of Potamotrygon magdalenae in the middle Magdalena River basin Four potential prey items were evaluated for their contributions to the P. magdalenae diet. According to mixing model estimates, in which we excluded 11 consumer signatures laying outside the 95% confidence intervals of the isotopic mixing polygon (Electronic Supplementary Material Table S2 and Fig. 3c), Coleoptera was the most important source assimilated by the species, with a percentage contribution ranging from 27.7 to 55.6%, followed by Ephemeroptera, which contributed highly variable amounts to the overall diet (3.6–55.2%) (Fig. 3b). In contrast, Diptera (Chironomidae) and Trichoptera contributed little to the P. magdalenae diet (0.4– 39.4% and 0.4–34.2%, respectively; Fig. 3b). Isotopic niche breadth (SEAB) based on the standard ellipse area showed differences among trophic guilds. The migratory detritivore Prochilodus magdalenae (Steindachner, 1879) (Pro) displayed the largest isotopic niche, and the carnivore Megalonema xanthum Eigenmann 1912 (Mxa) and the insectivore/invertivore Spatuloricaria gymnogaster (Eigenmann and Vance 1912) (Sgy) had the lowest values, with Potamotrygon Environ Biol Fish Table 2 Means and standard deviations (SD) of carbon isotope ratios (δ13C and δ15N) of Potamotrygon magdalenae between hydrological seasons in the middle Magdalena River basin. The sex and disc width (DW) is indicated. Bayesian standard ellipse areas (SEAB), parameters (eccentricity [E] and the angle between Season Sex n DW (Mean ± SD) δ13C (%o) δ13C Range %o the semi-major axis of the SEAc and the x-axis [θ]) and the area overlap (AO) of Bayesian standard ellipse areas of females and males of Potamotrygon magdalenae by hydrological seasons is also indicated δ15N (%o) (Mean ± SD) δ15N (Mean ± SD) SEAc SEAB E Range %o θ CI95% AO Dry F M 6 14.05 ± 4.26 6 13.87 ± 2.66 −29.12: − 21.52 −25.85 ± 2.80 −29.53: −21.89 −25.49 ± 3.14 10.14–12.39 10.84 ± 0.88 10.79–12.00 11.19 ± 0.53 6.91 3.55 5.34 3.26 0.97 13.17 2.35–14.37 0.80 0.99 8.02 1.30–8.83 Rainy F M 6 14.32 ± 2.87 6 14.22 ± 2.22 −27.83: −24.32 −28.95: −23.88 −26.43 ± 1.16 −26.76 ± 2.01 10.55–11.20 10.58 ± 0.39 10.21–11.38 10.70 ± 0.39 0.49 2.58 0.74 2.14 0.99 17.76 0.32–1.89 0.98 9.16 0.81–5.29 overlap, suggesting similar ecological roles. In comparison to other species, P. magdalenae (Pma) showed isotopic niche overlap with the omnivorous species magdalenae (Pma) also exhibiting low values (Fig. 4, Table 4). P. magdalenae (Pma) and Centrochir crocodili (Humboldt, 1821) (Ccr) exhibited high isotopic niche Dry season Rainy season 10 * 5 * * * 14 Females Males 80 60 Standard Ellipse Area (‰2) 15 100 b AEEb * AEEc Prey contribution to diet a 0.04 40 20 0 16 c Chi Col Eph Tri 0 Females Males 14 δ 15N δ 15N 13 12 11 12 10 10 8 9 8 6 -30 -28 -26 -24 -22 δ13C Fig. 3 a Isotopic niche space of females and males of Potamotrygon magdalenae by hydrological seasons presented as Bayesian ellipses. b. Boxplots depicting the relative isotopic contribution of prey items to Potamotrygon magdalenae in the middle Magdalena river basin. Sources include Coleoptera (Col), Ephemeroptera (Eph), Chironomidae (Chi) and Trichoptera (Tri). (Prey symbols are courtesy of Integration and Application Network, -30 -28 -26 -24 -22 -20 δ13C University of Maryland Center for Environmental Scienceian.umces.edu/symbols/) (c). Biplot showing the 13C and 15N signatures of the Potamotrygon magdalenae consumers inside (gray dots) and outside (white dots) of 95% mixing region (gray contour) and the average source signatures (black dots) with its standard deviation (dotted line) are shown Environ Biol Fish Table 3 Means and standard deviations (SD) of carbon and nitrogen isotope ratios (δ13C, δ15N) of Potamotrygon magdalenae, possible prey sources and the associated fish fauna in the middle Magdalena River basin. The trophic position (TP) also is indicated δ13C Species n Min δ15N Max Media SD Min Max Media SD TP Insectivores Elasmobranch Potamotrygon magdalenae 24 −29.53 −21.52 −26.13 2.30 9.97 12.39 10.83 0.60 3.31 Females 12 −29.12 −21.52 −26.14 2.1 9.97 12.39 10.713 0.7 3.27 Males 12 −29.53 −21.9 −26.13 2.6 10.2 12.01 10.945 0.5 3.36 6 −22.82 −22.07 −22.43 0.27 10.8 12.98 11.76 0.89 3.68 Teleostei Insectivores/Invertivores Spatuloricaria gymnogaster Detritivores Prochilodus magdalenae 6 −38.18 −22.9 −29.60 5.69 5.55 10.25 7.76 1.99 2.10 Curimata mivartii 6 −33.67 −28.43 −31.63 2.15 7.99 10.22 9.05 0.93 2.61 2.58 Omnivores Pimelodus blochii 6 −31.53 −22.38 −27.47 3.19 7.59 10.61 8.95 1.15 Sternopygus aequilabiatus 5 −28.73 −21.59 −24.54 2.82 9.48 11.35 10.55 0.75 3.20 Centrochir crocodili 6 −29.44 −24.92 −26.83 2.17 9.92 11.33 10.69 0.61 3.26 Sorubim cuspicaudus 6 −26.41 −22.67 −24.95 1.44 9.68 12.21 11.12 1.00 3.43 Omnivores/Herbivores 6 −28.17 −22.88 −25.36 2.34 10.50 12.42 11.42 0.72 3.55 2.62 Leporinus muyscorum Carnivores Triportheus magdalenae 6 −23.42 −18.24 −21.17 1.78 7.93 9.86 9.07 0.64 Astyanax magdalenae 6 −25.06 −18.04 −22.90 2.56 6.82 11.27 9.26 1.49 2.70 Pimelodus grosskopfii 6 −27.59 −19.97 −23.03 2.60 9.38 11.09 10.28 0.71 3.10 3.15 Trachelyopterus insignis 6 −24.10 −22.13 −23.29 0.76 9.36 11.61 10.40 0.89 Megalonema xanthum 6 −24.85 −22.42 −23.42 0.95 10.8 11.39 11.09 0.30 3.42 Cynopotamus magdalenae 6 −29.93 −25.24 −26.69 1.89 11.2 12.43 11.66 0.53 3.64 Carnivores/Piscivores 6 −25.45 −21.19 −23.37 1.61 10.9 12.51 11.66 0.55 3.64 8 −27.11 −23.64 −25.74 1.25 10.2 12.49 11.65 0.65 3.64 Chironomidae 2 −23.90 −23.17 −23.54 0.52 11.17 11.43 11.30 0.18 2.50 Ephemeroptera 5 −25.72 −24.85 −25.31 0.32 9.85 10.71 10.35 0.36 2.13 Coleoptera 11 −28.04 −19.87 −25.72 2.52 4.12 7.50 5.70 1.00 0.30 Trichoptera 4 −24.86 −23.34 −24.13 0.80 5.75 11.49 8.63 3.26 1.45 Pseudopimelodus bufonius Pseudoplatystoma magdaleniatum Insecta Sternopygus aequilabiatus (Humboldt, 1805) (Sae) and Sorubim cuspicaudus (Littmann et al. 2000) (Scu) and with the omnivore/herbivore Leporinus muyscorum (Steindachner 1900) (Lmu), while no overlap was identified with any detritivorous, carnivorous or carnivorous/piscivorous species (Fig. 4). The trophic position was intermediate for P. magdalenae, varying relatively little among females and males. Additionally, the trophic position was higher for the insectivore/invertivore Spatuloricaria gymnogaster and lower for the detritivore Prochilodus magdalenae (Table 3). Environ Biol Fish 80 CMA LMU PSM SCU PSB SGY MXA Trophic guilds CCR 11 PMA SAE Car TIN PGR 60 Car-Pis AMA Standard Ellipse Area (‰2) δ 15N 9 TMA PBL CMI Det Ins Omn Omn-Her PRO 40 7 -35 -30 -25 20 -20 δ13C 0 MXA SGY TIN PSM PSB CCR PMA CMA TMA SCU PGR AMA LMU CMI SAE PBL PRO Species Fig. 4 Bayesian estimates of the standard ellipse area (SEAB) of Potamotrygon magdalenae and the associated fish fauna in the middle Magdalena River basin. Box inside: Isotopic niche space of Potamotrygon magdalenae and bony fishes presented as Bayesian ellipses. The trophic groups of each species are indicated in colors, Green: Detritivores, Light Blue: Insectivores, Blue: Omnivores, Purple: Omnivores-Herbivores, Orange: Carnivores, Yellow: Carnivores-Piscivores. Species abbreviations are defined in Table 4 Table 4 Bayesian standard ellipse areas (SEAB) of Potamotrygon magdalenae and the associated fish fauna in the middle Magdalena River basin. Species abbreviations are indicated Species Abbreviation SEAC SEAB IC 95% Potamotrygon magdalenae Pma 3.09 2.95 1.99–4.53 Pseudopimelodus bufonius Psb 3.39 2.48 0.90–6.50 Pseudoplatystoma magdaleniatum Psm 2.35 2.02 0.93–4.40 1.57–10.09 Pimelodus grosskopfii Pgr 4.71 3.76 Pimelodus blochii Pbl 12.04 9.63 3.31–23.37 Megalonema xanthum Mxa 0.64 0.51 0.25–1.57 Sorubim cuspicaudus Scu 4.55 3.64 1.47–9.14 Spatuloricaria gymnogaster Sgy 0.88 0.70 0.27–1.68 Trachelyopterus insignis Tin 2.51 2.01 0.71–4.71 Centrochir crocodili Ccr 3.54 2.83 1.21–7.57 Leporinus muyscorum Lmu 6.60 5.28 1.84–12.42 Sternopygus aequilabiatus Sae 7.85 5.89 1.70–15.77 Astyanax magdalenae Ama 6.22 4.97 2.86–18.03 Cynopotamus magdalenae Cma 3.75 3.00 1.19–7.18 Curimata mivartii Cmi 7.08 5.66 2.02–13.20 Prochilodus magdalenae Pro 39.75 31.79 8.65–75.02 Triportheus magdalenae Tma 4.21 3.37 1.23–7.94 Environ Biol Fish Discussion The stomach content analysis indicated that P. magdalenae is a specialist predator that feeds on insects, mainly Chironomidae and Ceratopogonidae larvae, with some allochthonous prey consumed in low proportions. Feeding studies of congeneric species, such as Potamotrygon signata, P. orbignyi, and P. motoro, have shown an essentially insectivorous feeding habit with high consumption of Diptera larvae, Ephemeroptera nymphs and Odonata (Silva and Uieda 2007; Shibuya et al. 2009; Moro et al. 2011, 2012). Even among species that exhibit feeding plasticity, such as Potamotrygon falkneri (Castex and Maciel 1963) and P. motoro, which prey upon mollusks, crustaceans and fishes at some stages of their life history, insects are also an important food source (Lonardoni et al. 2006; Silva and Uieda 2007; Lasso et al. 2013). This similarity in the food preferences of Potamotrygon species, as in other taxonomic groups, has been suggested to be an evolutionary strategy for avoiding strong competition for food resources with other groups (Wiens and Graham 2005; Losos 2008). Likewise, larval insects are important food sources for many freshwater fishes, mainly stream-dwelling species (e.g., Moreira and Zuanon 2002; Catarino and Zuanon 2010), being consumed during different life stages and at different proportions and being often abundant in the Neotropical region (Borkent and Spinelli 2007; Ferrington 2008), including Colombian rivers (e.g., Ramírez and Pringle 1998; Roldán et al. 2014). Although the results indicate a tendency toward dietary specialization in P. magdalenae, which is not widespread among elasmobranch species (Wetherbee et al. 2012), and although no quantitative biomass information is available regarding the prey species in the study area, the high consumption of an abundant group of prey, such as Diptera, suggests an opportunistic foraging mode, as mentioned for other potamotrygonids (e.g., Lonardoni et al. 2006; Gama and Rosa 2015). Furthermore, due to the large variability in habitats and resources in tropical fluvial ecosystems, freshwater fish species are generally considered mostly opportunistic in their feeding habits (Lowe-McConnell 1987). In a previous study conducted on P. magdalenae in the higher portion of the Magdalena River, RamosSocha and Grijalba-Bendeck (2011) reported high consumption of Polymitarcyidae and their larvae, which are very abundant prey in this area. In this regard, it seems that the feeding habits of P. magdalenae could be framed within the optimal foraging theory, which predicts that consumers should select prey items to optimize their energy intake in relation to the costs of catching, ingesting and digesting these prey items (Pyke et al. 1977). Diet composition and isotopic analysis did not show significant differences between males and females, suggesting they could share the same trophic niche and feed in the same habitats and on the same prey or prey of similar trophic levels. Likewise, we observed similar isotopic signals between seasons for females and males. However, the slight depletion of carbon isotopic signals in the rainy season may indicate a seasonal shift in the basal carbon source that could not be fully captured by isotopic analysis. Therefore, the carbon signals of fishes in tropical rivers have been identified as being depleted during the wet season, suggesting that during this period, fish biomass appears to be derived mostly from C3 macrophytes, while that during the dry season appears to be derived mostly from algae (e.g., Forsberg et al. 1993; Jepsen and Winemiller 2007; Ou and Winemiller 2016). For a more detailed assessment of seasonal shifts in isotopic signals in freshwater stingrays, we suggest the use of liver tissue, which has higher rates of isotopic incorporation than muscle tissue and could exhibit the food sources consumed over several weeks (MacNeil et al. 2005, 2006; Logan and Lutcavage 2010). Examination of isotopic niches using standard ellipse areas and their parameters suggest that males and females of P. magdalenae tend to exploit food resources of similar trophic positions but from different carbon sources. This is supported by the fact that many of the insects consumed by P. magdalenae use organic matter (of aquatic and terrestrial origin) as their main food source (Nessimian and Sanseverino 1998), which may result in a slightly wide range of δ13C values. Although a larger isotopic niche area was observed for females, it is possible that the small sample sizes for these analyzed categories influenced the isotopic estimate. The isotopic niche overlap between males and females was higher in the dry season, when the isotopic niches were larger, and was reduced in the rainy season. This similarity suggests that the individuals exploited similar food resources within the same environment and that these preys possibly used a wider range of carbon sources than in the rainy season. As discussed by some authors (Goulding 1980; Prejs and Prejs 1987; Matthews 1998), increased dietary overlap can occur Environ Biol Fish when food becomes limited, and thus predators become less selective (Blaber 1986). In contrast, it can also occur as a result of an increased abundance of prey items (Thorburn et al. 2014). The results of this study may be related to the latter pattern, as supported by the highest abundance of aquatic insects during the dry season and the transition to the rainy season in this region (Arias-Díaz et al. 2007; Forero-Céspedes et al. 2016). This pattern of dietary overlap can occur depending on the characteristics of the system, such as its productivity, the nature of the resources (e.g., autochthonous vs. allochthonous) and the characteristics of the species (e.g., generalist vs. specialist). The stable isotope analysis in this study indicated that the food resource most frequently reported in stomach content analysis may not be the most energetically important prey item for P. magdalenae. Our results showed that while Chironomidae were observed to be particularly important in the diet of P. magdalenae, stable isotope analysis revealed that Coleoptera and Ephemeroptera were the most energetically important food sources for the species. The interpretation of consumer assimilation of production sources depends, in part, on isotopic turnover rates in tissues. For P. motoro, the turnover rate of muscle was estimated to be 422 days (MacNeil et al. 2006), and assuming a similar turnover rate for P. magdalenae, it is expected that these results reflect the consumed and assimilated food sources 1 year (or more) before the sampling. However, it must be considered that turnover is influenced by taxon, tissue type, life stage, environment and other factors (Kim et al. 2012). Therefore, these results suggest that species vary in prey consumption, reflecting the natural abundance of prey in different seasons. In this regard, many tropical insect taxa undergo seasonal changes in abundance in response to the wet and dry seasons and the available resources (Wolda 1980). For example, some authors argue that environmental stability in the winter dry season can assure a high availability of insect larvae (Huamantinco and Nessimian 1999). The highest abundance of aquatic Coleoptera and Ephemeroptera in rivers of the Magdalena basin occurred during the dry period and in the transition to the rainy season (Arias-Díaz et al. 2007; Forero-Céspedes et al. 2016). Furthermore, isotopic signals of eleven consumers could not be explained by the source data (See Mixing polygon, Electronic Supplementary Material Table S2 and Fig. 3c), suggesting that other important food sources may exist for P. magdalenae that were not collected and analyzed. Many of these consumers were heavily 13C-depleted, likely by feeding on prey that consume organic matter from C3 vegetation, such as some detritivorous aquatic insects (Davis et al. 2012; Ou and Winemiller 2016; Jardine et al. 2017). Stomach content and stable isotope analyses corroborated the trophic level of P. magdalenae. For several species of Potamotrygonidae, intermediate trophic levels have been reported, identifying them as secondary consumers in river food webs (Jacobsen and Bennett 2013). Overall, batoid fishes are considered mesopredators that provide important links between the lower trophic levels and top predators (Vaudo and Heithaus 2011). Although it cannot be considered a top predator due to its intermediate trophic level, P. magdalenae may have an exclusive predator role in the Magdalene River ecosystem, as no species have been reported to prey upon this species. Nevertheless, predation of Potamotrygon spp. by the giant otter Pteronura brasiliensis (Zimmerman 1780) was reported in the Orinoco River basin (Gómez-Serrano 2004); thus, the otter present in the Magdalena River [Lontra longicaudis (Olfers, 1818)] could be considered a potential predator of P. magdalenae. If this trophic relationship is confirmed, P. magdalenae would be supported as being a mesopredator and as a link of energy transfer between low and high trophic levels, similar to marine batoid species (Navia et al. 2017). P. magdalenae and many of the analyzed fish species were not clearly differentiated in the isotopic niche. This mixing of carbon and nitrogen sources is the result of the complex spatiotemporal dynamics of tropical lowland rivers, the diversity of terrestrial and aquatic food sources available to consumers at any given time and the variable degree of opportunistic feeding habits of freshwater fishes (Goulding 1980). Thus, it is difficult to link fluxes in food resources with consumer population dynamics (Jepsen and Winemiller 2002). However, differences between morphologic and trophic diversity within freshwater fishes can provide insight into how the species may be partitioning the niche space. For example, P. magdalenae and the other insectivore species S. gymnogaster were differentiated in terms of isotopic niche, with the latter exhibiting a more enriched carbon signal, suggesting the consumption of prey from different carbon sources or space segregation for feeding activities. The enrichment with insect protein for the genus Spatuloricaria (Melo et al. 2004), which may correspond with specialized morphologies Environ Biol Fish or behaviors, likely contributes to its elevated δ15N and the possible assimilation of more 15N-enriched seston resources (Lujan et al. 2011). On the other hand, P. magdalenae could be considered redundant in its isotopic space with some omnivorous species, such as Sternopygus aequilabiatus (Humboldt, 1805) (Sae) and Sorubim cuspicaudus, which also include insects in their diets but also feed on seeds and crustaceans (e.g., Agualimpia et al. 2007). These results suggest the consumption of prey with similar carbon sources and trophic levels. In this context, differences in habitat use or source exploitation could decrease their interspecific interactions within a habitat. For example, in floodplain lakes in the Magdalena River basin, an omnivorous species, the thorny catfish (Centrochir crocodili) and S. aequilabiatus are more active at night, while P. magdalenae is more active during the day (Hernández-Serna et al. 2015). The catfish Pseudoplatystoma magdaleniatum (Buitrago-Suárez and Burr 2007) (Psm) and Pseudopimelodus bufonius (Valenciennes, 1840) (Psb) are carnivorous species that feed mainly on fishes (Maldonado-Ocampo et al. 2005; Buitrago-Suárez 2006) and whose consistently high δ15N values contribute to their high trophic levels. Finally, the detritivorous species Prochilodus magdalenae and Curimata mivartii (Steindachner, 1878) (Cmi) display greater variation in their isotopic niches. Detritivorous prochilodontids are bottom feeders, possess fleshy lips and tiny teeth that likely aid in dislodging flocculent material, and assimilate large fractions of organic matter associated with algae and microorganisms derived from the substratum (Goulding 1980; Ou and Winemiller 2016). This large variation can be the result of feeding from different food webs in this aquatic environment, considering that those species undergo annual long-distance migrations (Jiménez-Segura et al. 2016). As a final point, P. magdalenae could be considered to be an important predator of aquatic insects in this system, especially based on the low trophic redundancy among other species that feed on insects, their higher rates of consumption of these resources, and their lower natural abundance versus that of other insectivorous and omnivorous species. Accordingly, it is still necessary to determine whether this trophic relationship is influencing the abundance of these resources and is consequently an important structuring force in the community. This study is a first glimpse of the ecological role of the endemic freshwater stingray P. magdalenae in the Magdalena River basin, providing insight for future studies to determine how the different species may be partitioning niche space and how they respond to the main threats in the Andean river basins in terms of resource utilization. Therefore, future studies regarding the ecological role of P. magdalenae and other predators in this area should attempt to incorporate spatial and temporal scales that will allow us to explore how they alter these roles in response to the community dynamics of fishes and other food sources, which are highly synchronized with seasonality and fishing pressure. Acknowledgments VMV thanks the Postgraduate Student Agreement Program (PEC-PG) and the National Council for Scientific and Technological Development, Brazil (CNPq) (Proc. 190513/2014-4), for the master scholarship. Thanks are also due to the fishermen of La Pesca, Antioquia, Colombia, for their valuable contributions to the collection of samples and to colleagues of the SQUALUS Foundation, especially J. López, for their assistance conducting laboratory work and discussions regarding the statistical analysis of the isotope data. This study was supported financially by the Rufford Foundation (RSG-18238-1). Author contributions AFN and VMV conceived and designed the study. VMV collected data, compiled data from the literature and performed the laboratory work. AFN contributed materials, reagents, and analytical tools. VMV, EG and AFN analyzed the data. VMV, RSR, EG and AFN contributed to the interpretation of the results. 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